Brucella epitope recognition by CD8+ T cells

描述（由申请人提供）：布鲁氏菌是一种细胞内的人类病原体，主要由CD8+ T细胞控制，但含有被保护性CD8+ T细胞识别的肽的布鲁氏菌蛋白尚不清楚。我们的目标是鉴定特异性布鲁氏菌CD8+ T细胞表位，并测试它们保护小鼠免受致病性布鲁氏菌感染的能力。为此，我们将首先分离并鉴定感染巨噬细胞上与主要组织相容性复合体I类（MHC I）分子结合的自然加工和呈递的布鲁氏菌特异性T细胞表位。T细胞表位将被鉴定使用质谱（MS）和生物信息学相关联的表位与各自的布鲁氏菌蛋白。其次，我们将利用侵入性大肠杆菌载体对小鼠进行免疫，评估CTL对布鲁氏菌抗原的活性。第三，我们将评估抗原性布鲁氏菌蛋白和T细胞表位对小鼠清除毒力布鲁氏菌的影响。我们有信心根据初步数据实现这些目标，这些数据表明我们有能力实现每一个具体目标。此外，我们还显示了重组大肠杆菌载体免疫小鼠对抗原的显著CTL反应。我们相信，我们的目标是开发和测试这种新的方法来阐明CD8+ CTL表位及其在体内清除布鲁氏菌的作用，这将填补在鉴定布鲁氏菌蛋白方面的空白，这些蛋白对细胞毒性CD8+ T细胞清除这种细胞内细菌很重要。我们的具体目标是：1。鉴定自然加工和MHC I呈现的布鲁氏菌肽。小鼠巨噬细胞RAW264.7细胞感染布鲁氏菌。MHC i -布鲁氏菌肽复合物将从细胞膜上共免疫沉淀并纯化肽。多肽将进行LC-MS分析，数据将分析布鲁氏菌T细胞表位。布鲁氏菌肽结合MHC I分子的亲和力将用于确认高结合肽的鉴定。2. 评估CTL对含有特定T细胞表位的布鲁氏菌蛋白的活性。侵入性大肠杆菌载体将被改造以表达含有T细胞表位的布鲁氏菌蛋白。这些重组大肠杆菌载体将用于小鼠免疫。表达布鲁氏菌肽的H-2单倍型匹配细胞系将被用作刺激/靶细胞，用于免疫小鼠脾t细胞的CTL检测。CTL数据分析将鉴定含有CD8+ T细胞表位的布鲁氏菌蛋白，这对清除细胞内布鲁氏菌很重要。评估侵入性大肠杆菌载体传递的布鲁氏菌蛋白诱导CTL活性和宿主保护作用。用表达布鲁氏菌蛋白的侵袭性大肠杆菌载体免疫小鼠，可测定表达luxcabe的毒力性梅利特氏杆菌的清除率，清除率与CTL活性相关。将布鲁氏菌特异性CD8+ T细胞过继转移到幼稚受体小鼠身上，将证实不同的布鲁氏菌特异性ctl清除毒力贝氏杆菌感染的有效性。因此，这一目的将CTL活性与清除布鲁氏菌感染的生物学相关性联系起来。鉴定含有CD8+ T细胞表位的布鲁氏菌蛋白对于理解布鲁氏菌蛋白质组对免疫识别和宿主保护的贡献至关重要。项目描述：该提案将确定特定的B. melitensis CD8+ T细胞表位，并测试它们保护小鼠免受毒力布鲁氏菌感染的能力。我们将首先分离并鉴定感染巨噬细胞上与主要组织相容性复合体I类（MHC I）分子结合的自然加工和呈递的布鲁氏菌特异性T细胞表位。T细胞表位将被鉴定使用质谱（MS）和生物信息学相关联的表位与各自的布鲁氏菌蛋白。其次，我们将利用侵入性大肠杆菌载体对小鼠进行免疫，评估CTL对布鲁氏菌抗原的活性。第三，我们将利用体内生物光子成像技术评估用含有T细胞表位的抗原性布鲁氏菌蛋白免疫小鼠清除小鼠体内毒性布鲁氏菌的影响。我们有信心根据初步数据实现这些目标，这些数据表明我们有能力实现每一个具体目标。

DESCRIPTION (provided by applicant): Brucella melitensis, an intracellular human pathogen, is largely controlled by CD8+ T cells, but the Brucella proteins containing peptides recognized by protective CD8+ T cells remain unknown. Our goal is to identify specific Brucella CD8+ T cell epitopes and test their ability to protect mice against infection with virulent Brucella. To do this we will first isolate and identify naturally processed and presented Brucella-specific T cell epitopes bound to Major Histocompatibility Complex class I (MHC I) molecules on infected macrophages. T cell epitopes will be identified using Mass Spectrometry (MS) and bioinformatics to correlate an epitope with the respective Brucella protein. Second, we will assess CTL activity against the Brucella antigens using an invasive E. coli vector to immunize mice. Third, we will assess the impact of antigenic Brucella proteins and T cell epitopes on clearance of virulent Brucella from mice. We are confident of achieving these goals based on preliminary data that demonstrate our ability in each specific aim. Also, we have shown significant CTL response to antigen from mice immunized with the recombinant E.coli vectors. We believe our goal of developing and testing this novel approach to elucidate CD8+ CTL epitopes and their contributions to clearance of Brucella in vivo will fill the void in identifying Brucella proteins important for clearance of this intracellular bacterium by cytotoxic CD8+ T cells. Our specific aims are: 1. Identify naturally processed and MHC I presented Brucella peptides. Mouse macrophage RAW264.7 cells will be infected with Brucella melitensis. MHC I-Brucella peptide complex will be co- immunoprecipitated from the cell membrane and the peptide purified. Peptides will then be subject to LC-MS, and data will be analyzed for Brucella T cell epitopes. Affinity of Brucella peptide binding to MHC I molecules will be used to confirm the identify of high binding peptides. 2. Assess CTL activity against Brucella proteins containing specific T cell epitopes. Invasive E. coli vectors will be engineered to express Brucella proteins containing T cell epitopes. These recombinant E. coli vectors will be used to immunize mice. H-2 haplotype matched cell lines expressing the Brucella peptides will be used as stimulator/target cells for CTL assays of splenic T-cells isolated from the immunized mice. CTL data analysis will identify Brucella proteins containing CD8+ T cell epitopes important for clearance of intracellular Brucella. 3. Evaluate Brucella proteins delivered by invasive E. coli vectors to induce CTL activity and host protection. Clearance of luxCDABE-expressing virulent B. melitensis will be determined in mice immunized with invasive E. coli vectors expressing Brucella proteins and clearance will be correlated to CTL activity. Adoptive transfer of Brucella specific CD8+ T cells to naive recipient mice will confirm the effectiveness of different Brucella-specific CTLs to clear virulent B. melitensis infection. Thus, this aim will link CTL activity to biologic relevance in clearing Brucella infection. Identifying Brucella proteins that contain CD8+ T cell epitopes would be crucial to understanding the contribution of the Brucella proteome to immune recognition and host protection. Project Narrative: The proposal will identify specific B. melitensis CD8+ T cell epitopes and test their ability to protect mice against infection with virulent Brucella. We will first isolate and identify naturally processed and presented Brucella-specific T cell epitopes bound to Major Histocompatibility Complex class I (MHC I) molecules on infected macrophages. T cell epitopes will be identified using Mass Spectrometry (MS) and bioinformatics to correlate an epitope with the respective Brucella protein. Second, we will assess CTL activity against the Brucella antigens using an invasive E. coli vector to immunize mice. Third, we will assess the impact of mice immunized with antigenic Brucella proteins containing T cell epitopes to clear virulent Brucella from mice using in vivo biophotonic imaging. We are confident of achieving these goals based on preliminary data that demonstrate our ability in each specific aim. The Problem: Brucella melitensis, a Gram-negative facultative intracellular bacterium, induces chronic infectious disease in humans and domestic animals. Immunological protection against intracellular Brucella appears to be mediated by cytotoxic CD8+ T cells, but the peptides recognized by protective cytotoxic T cells remain unknown. Identifying Brucella proteins that contain CD8+ T cell epitopes would be crucial to understanding the contribution of the Brucella proteome to immune recognition. What Brucella genes or proteins should encompass an effective, safe Brucella vaccine for humans is poorly understood. The Product: Our goal of developing and testing this novel approach to elucidate CD8+ CTL epitopes and their contributions to clearance of Brucella in vivo will fill the void in identifying Brucella proteins important for clearance of this intracellular bacterium by cytotoxic CD8+ T cells. The invasive, non- pathogenic E. coli vector can stimulate a potent CTL response providing a strategy for Brucella immunization to test identified MHC I associated epitopes for CD8+ T cell protective immunization. Identifying B. meltinesis peptides that confer protection and determining the mechanisms that initiate host protection will permit designing vaccine vector delivery systems for host protection.